Maxwell 16 automated purification system

Two weeks after seeding, the youngest fully expanded leaves of O. sativa were frozen in liquid nitrogen and stored at -80 °C prior to RNA isolation for RNA-Seq. Frozen samples were homogenized with zirconia beads YTZ-4 (AS-ONE, Osaka, Japan) using TissueLyser II (Qiagen, Hilden, Germany), and total RNA was extracted using the Maxwell 16 LEV Plant RNA Kit (Promega, Madison, WI, USA) and the Maxwell 16 Automated Purification System (Promega). The concentration of RNA was measured using a QuantiFluor RNA System (Promega) and Quantus Fluorometer (Promega, Madison, WI, USA).
Seven days after seeding, bulked seedlings of A. thaliana were homogenized with zirconia beads YTZ-4 using a TissueLyser II (Qiagen, Hilden, Germany), and total RNA was extracted using the Maxwell 16 LEV Plant RNA Kit (Promega, Madison, WI, USA) and the Maxwell 16 Automated Purification System (Promega, Madison, WI, USA). The RNA concentration was measured using a Quantus Fluorometer (Promega, Madison, WI, USA).

RNA was extracted using a Maxwell^® 16 Automated Purification System (Promega, Madison, WI, USA) with Fruit-mate™ for RNA Purification (Takara Bio, Kusatsu, Japan). The RNA concentration was determined with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and stored at −80 °C.
For reverse transcription we used 100 ng total RNA with the ReverTra Ace^® qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s protocol. The cDNA solution was diluted two-fold and stored at −20 °C.
Real-time PCR was carried out in a LightCycler Nano or 480 (Roche Diagnostics, Basel, Switzerland) with the THUNDERBIRD^® SYBR^® qPCR Mix (Toyobo). The PCR assay was prepared to 20 µL total volume containing 10 µL THUNDERBIRD^® SYBR^® qPCR Mix, 2.0 µL cDNA solution, and 10 µM gene-specific primers. The gene-specific primer sequences are listed in Table S1. The thermal cycling procedure was as follows: 95 °C for 30 s, three-step amplification consisting of 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, a pre-melt hold at 95°C for 10 s, and melting at 60 °C to 97 °C at a rate of 0.1 °C/s. All target gene data were normalized against EF1α transcript levels.

RNA was extracted automatically with a Maxwell 16 Automated Purification system (Promega, Madison, WI, USA), as described by Ishibashi et al. [43 (link)]. As a pre-treatment, 50–100 mg of frozen powdered sample was mixed with 500 µL of Fruit-mate (Takara Bio, Kusatsu, Japan). The mixture was vortexed and centrifuged at 13,000× g for 5 min. To 400 µL of supernatant, 200 µL lysis buffer was added to prepare the sample solution for extraction using the Maxwell purification system “RNA-PLANT” protocol. The resulting RNA samples were stored at –80 °C.